Solution Equilibrium Studies on Salicylidene Aminoguanidine Schiff Base Metal Complexes: Impact of the Hybridization with L-Proline on Stability, Redox Activity and Cytotoxicity

The proton dissociation processes of two tridentate salicylidene aminoguanidine Schiff bases (SISC, Pro-SISC-Me), the solution stability and electrochemical properties of their Cu(II), Fe(II) and Fe(III) complexes were characterized using pH-potentiometry, cyclic voltammetry and UV-visible, 1H NMR and electron paramagnetic resonance spectroscopic methods. The structure of the proline derivative (Pro-SISC-Me) was determined by X-ray crystallography. The conjugation of L-proline to the simplest salicylidene aminoguanidine Schiff base (SISC) increased the water solubility due to its zwitterionic structure in a wide pH range. The formation of mono complexes with both ligands was found in the case of Cu(II) and Fe(II), while bis complexes were also formed with Fe(III). In the complexes these tridentate ligands coordinate via the phenolato O, azomethine N and the amidine N, except the complex [Fe(III)L2]+ of Pro-SISC-Me in which the (O,N) donor atoms of the proline moiety are coordinated beside the phenolato O, confirmed by single crystal X-ray crystallographic analysis. This binding mode yielded a stronger Fe(III) preference for Pro-SISC-Me over Fe(II) in comparison to SISC. This finding is also reflected in the lower redox potential value of the iron-Pro-SISC-Me complexes. The ligands alone were not cytotoxic against human colon cancer cell lines, while complexation of SISC with Cu(II) resulted in moderate activity, unlike the case of its more hydrophilic counterpart.


Introduction
Aminoguanidine (also known as pimagedine, Scheme 1) is an α,β-dicarbonyl scavenging agent and was developed for the treatment of diabetic kidney disease, due to its ability to reduce the levels of advanced glycation end products which manages diabetic nephropathy [1]. The guanidine fragment appears in proteins as it is found in the side chain of arginine, and is generally positively charged at physiological pH; however, the guanidinium pK a can be varied by the linked molecular moieties. Aminoguanidine can form Schiff bases with aldehydes or ketones, both in vivo (e.g., with pyridoxal phosphate [2]), and in vitro as reported in numerous papers [3][4][5][6][7]. Schiff bases (imines) and their transition metal complexes are widely investigated and considered as key classes of medicinal compounds due to their enormous potential for pharmacological activities such as anticancer, medicinal compounds due to their enormous potential for pharmacological activities such as anticancer, antibacterial and antioxidant effects [8,9]. Among the metal complexes of aminoguanidine-based Schiff bases, the Cu(II) complexes formed with salicylidene aminoguanidine or pyridoxilidene aminoguanidine are the most common [3,4,6,10,11], although we can find examples for V(V), Co(III) or Ni(II) complexes as well [5,6,10,12,13]. The Cu(II) complex of the (N,N,O) donor containing a Schiff base of aminoguanidine formed with salicylaldehyde (2-(2-hydroxybenzylidene)hydrazinecarboximidamide, SISC, Scheme 1) displayed significant cytotoxic activity on a colorectal cancer cell line (HCT1) and strong DNA-binding affinity [3]. The Cu(II) complex formed with the (N,N,N) donor containing a Schiff base of aminoguanidine and 2-acetylpyridine showed moderate and slight activity toward bacteria and toward molds, respectively [6].  [14][15][16]. Triapine (Scheme 1) is the best-known representative of TSCs, which has been studied in ca. 30 phase I and II clinical trials as an anticancer drug [15,17], and is ready to enter phase III clinical trials [18]. Its activity is linked to the inhibition of the ribonucleotide reductase enzyme based on its complex formation ability with iron ions [19]. In our previous work, the significant effect of the variation of the chalcogen atom in the TSC scaffold from sulfur to oxygen (semicarbazone) or selenium (selenosemicarbazone) on the pKa values and the Fe(II)-, Fe(III)-and Cu(II)-binding ability, in addition to the cytotoxicity, was reported [20]. It was found that the iron preference has a strong impact on the biological activity due to the suggested mechanism of action of terminally non-substituted TSCs such as Triapine (Scheme 1). e.g., the exchange of the thioamide sulfur to oxygen in Triapine resulted in a loss of cytotoxicity, most probably due to the inability of oxo-Triapine to bind iron ions efficiently in both oxidation states (+2 and +3). On the other hand, the Fe(III) preference of the salicylaldehyde TSC (STSC, Scheme 1) possessing (O,N,S) chelating moiety is also stronger over Fe(II) in comparison with the α-N-pyridyl TSCs (e.g., Triapine), which seems to be an unfavorable property in terms of the cytotoxic activity [21,22]. The salicylaldehyde semicarbazone (or salicylidene aminoguanidine, SSC, Scheme 1) with (O,N,O) donor set is also a non-cytotoxic compound [23]. Meanwhile, the Cu(II) complexes, both STSC and SSC, are reported to have moderate cytotoxicity [24][25][26]. Thiosemicarbazones (TSC) are also Schiff base ligands possessing numerous pharmacological applications, and the tridentate derivatives often coordinate via (N,N,S) or (O,N,S) donor sets [14][15][16]. Triapine (Scheme 1) is the best-known representative of TSCs, which has been studied in ca. 30 phase I and II clinical trials as an anticancer drug [15,17], and is ready to enter phase III clinical trials [18]. Its activity is linked to the inhibition of the ribonucleotide reductase enzyme based on its complex formation ability with iron ions [19]. In our previous work, the significant effect of the variation of the chalcogen atom in the TSC scaffold from sulfur to oxygen (semicarbazone) or selenium (selenosemicarbazone) on the pK a values and the Fe(II)-, Fe(III)-and Cu(II)-binding ability, in addition to the cytotoxicity, was reported [20]. It was found that the iron preference has a strong impact on the biological activity due to the suggested mechanism of action of terminally nonsubstituted TSCs such as Triapine (Scheme 1). e.g., the exchange of the thioamide sulfur to oxygen in Triapine resulted in a loss of cytotoxicity, most probably due to the inability of oxo-Triapine to bind iron ions efficiently in both oxidation states (+2 and +3). On the other hand, the Fe(III) preference of the salicylaldehyde TSC (STSC, Scheme 1) possessing (O,N,S) chelating moiety is also stronger over Fe(II) in comparison with the α-N-pyridyl TSCs (e.g., Triapine), which seems to be an unfavorable property in terms of the cytotoxic activity [21,22]. The salicylaldehyde semicarbazone (or salicylidene aminoguanidine, SSC, Scheme 1) with (O,N,O) donor set is also a non-cytotoxic compound [23]. Meanwhile, the Cu(II) complexes, both STSC and SSC, are reported to have moderate cytotoxicity [24][25][26].
In the present comparative study, SISC (Scheme 1), the aminoguanidine congener of STSC, was applied to investigate the effect of the =NH/=S exchange on the solution and biological properties. Additionally, its enantiomerically pure proline hybrid (R-1-(3-)((2carbamimidoylhydrazono)methyl)-2-hydroxy-5-methylbenzyl)pyrrolidine-2-carboxylic acid, Pro-SISC-Me, Scheme 1) with increased water solubility was also synthesized and characterized. Therefore, the proton dissociation processes and lipophilicity of SISC and Pro-SISC-Me, the solution stability, stoichiometry and electrochemical properties of their Cu(II), Fe(II) and Fe(III) complexes, were studied and compared to the analogous TSCs. The relationship between these data and the cytotoxicity of the compounds in Colo-205 (human colon cancer), Colo-320 (doxorubicin-resistant human colon cancer) and normal human embryonal lung fibroblast (MRC-5) cells was also examined.

Synthesis and Characetriazion of Pro-SISC-Me
The chiral compound Pro-SISC-Me·HCl·2H 2 O was prepared by condensation reaction of 2-hydroxy-3-methyl-(S)-pyrrolidine-2-carboxylate-5-methylbenzaldehyde with in situ prepared aminoguanidinium chloride in boiling methanol/water in 61% yield. The formation of the Schiff base was confirmed by elemental analysis, as well as by single crystal X-ray diffraction (SC-XRD) analysis of the aminoguanisonium bromide salt (Pro-SISC-Me·HBr (1)) obtained similarly to the chloride salt (Pro-SISC-Me·HCl·2H 2 O). The result of X-ray diffraction study of 1 is shown in Figure 1 with selected metric parameters quoted in the legend. Even though the series of transition metal complexes formed with TSCs derived from the same aldehyde or 2-formylpyridine coupled via a methylene group to L-proline moiety is well documented in the literature [27][28][29][30][31][32], there is only one structural characterization of a metal-free TSC [28].
In the present comparative study, SISC (Scheme 1), the aminoguanidine congener of STSC, was applied to investigate the effect of the =NH/=S exchange on the solution and biological properties. Additionally, its enantiomerically pure proline hybrid (R-1-(3-)((2-carbamimidoylhydrazono)methyl)-2-hydroxy-5-methylbenzyl)pyrrolidine-2carboxylic acid, Pro-SISC-Me, Scheme 1) with increased water solubility was also synthesized and characterized. Therefore, the proton dissociation processes and lipophilicity of SISC and Pro-SISC-Me, the solution stability, stoichiometry and electrochemical properties of their Cu(II), Fe(II) and Fe(III) complexes, were studied and compared to the analogous TSCs. The relationship between these data and the cytotoxicity of the compounds in Colo-205 (human colon cancer), Colo-320 (doxorubicin-resistant human colon cancer) and normal human embryonal lung fibroblast (MRC-5) cells was also examined.

Solution Chemistry of SISC and Pro-SISC-Me
The studied compounds (Scheme 1) have relatively high thermodynamic solubility at pH 7.4 (S 7.4~2 mM (SISC), S 7.4~1 0 mM (Pro-SISC-Me), T = 25 • C, 10 mM HEPES); however, their metal complexes possess worse solubility in water. Therefore, most of the solution equilibrium studies were performed in 30% (v/v) DMSO/H 2 O solvent mixture in this work. Meanwhile, the proton dissociation processes of the ligands were also monitored in aqueous solutions. The proton dissociation constants (pK a ) were determined by pHpotentiometric, UV-visible (UV-vis) and 1 H NMR spectroscopic titrations and are collected in Table 1. Two pK a values could be determined for SISC by pH-potentiometry, while for the proline hybrid an additional proton dissociation process (characterized by pK 1 ) was detected in the studied pH range (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The fundamental difference between the TSCs (-C=N-NH-C(=S)NH 2 ) and the SCs (-C=N-NH-C(=O)NH 2 ) is that the iminosemicarbazone moiety (-C=N-NH-C(=NH)NH 2 ) is a basic group resulting in a positive charge when it becomes protonated. The deprotonation of COOH does not result in spectral changes in the UV-vis wavelength range, thus pK a could not be determined by this method; c These pK a values belong to the major species (E isomer), while pK a = 7.55 ± 0.02 was determined for the minor (Z) isomer and no other pK a could be determined as spectral changes were not detected between pH 9.3 and 11.5.
UV-vis and 1 H NMR spectra were recorded at different pH values for both ligands. The UV-vis spectra of SISC (Figures 2 and S1) and Pro-SISC-Me ( Figure S2) reveal very similar, although not identical, spectral changes upon increasing the pH, which suggests that the same functional groups of the ligands deprotonate in each step. Taking into account the pH dependence of the absorbance values (Figures 2c, S1c and S2c), two deprotonation steps can be observed. The molar absorbance spectra of the ligand species in the different protonation states (H 2 L + , HL, L − ) can be seen in Figures 2b, S1b and S2b. These individual spectra show that both deprotonation steps are accompanied by a significant red shift, especially in the case of the HL → L − process. The increased λ max has often originated from a more extended conjugated π-electron system, which is typically observed for the deprotonation of phenolic OH groups [22,24]. Notably, the deprotonation of the nonchromophoric proline moieties in Pro-SISC-Me (COOH, N Pro H + ) is not expected to result in significant spectral changes. Thus, pK 1 of Pro-SISC-Me (which could be determined by pH-potentiometry) belongs to the carboxylic acid group. 1 H NMR spectra recorded for SISC in 30% (v/v) d 6 -DMSO/H 2 O ( Figure S3) clearly show two deprotonation steps and all the CH protons are sensitive to the changes of the pH. Similarly, the changes in the chemical shifts of the peaks of the aromatic CH and the CH=N protons indicate two steps in the 1 H NMR spectra of Pro-SISC-Me recorded in water (Figure 3a), thus two pK a values were calculated (pK 2 , pK 3 in Table 1). However, this is only true for the major species identified as the E isomer (Figure 3b), while for the other isomer (Z, Figure 3c) only one pK a (7.55 ± 0.02) could be determined. The E and Z geometric isomers are the consequence of the C=N double bond. A possible hydrogen bond between the deprotonated phenolato oxygen and the hydrazonic NH might result in an increased pK a of the aminoguanidinium moiety (>11.5) and decreased pK a of the OH. Based on the integrals of the CH=N peaks, the fraction of the E isomer is 86% at pH < 6.5, and it is decreased to 82% reaching pH 11.5; however, the appearance of further minor peaks at pH > 9. 8 indicates the presence of other species in low fraction. The same picture is seen when the changes of the CH 3 peaks are considered ( Figure S4), and only the peak of the CH-COOH proton was sensitive to the first deprotonation process at pH < 3, which confirms that pK 1 of Pro-SISC-Me applies to COOH. In the presence of 30% (v/v) DMSO only one type of isomer is present (most probably E), or there is a fast exchange process between the isomers (in the case of SISC only one type of isomer was observed in both media).  Table 1). However, this is only true for the major species identified as the E isomer (Figure 3b), while for the other isomer (Z, Figure 3c) only one pKa (7.55 ± 0.02) could be determined. The E and Z geometric isomers are the consequence of the C=N double bond. A possible hydrogen bond between the deprotonated phenolato oxygen and the hydrazonic NH might result in an increased pKa of the aminoguanidinium moiety (>11.5) and decreased pKa of the OH. Based on the integrals of the CH=N peaks, the fraction of the E isomer is 86% at pH < 6.5, and it is decreased to 82% reaching pH 11.5; however, the appearance of further minor peaks at pH > 9. 8 indicates the presence of other species in low fraction. The same picture is seen when the changes of the CH3 peaks are considered ( Figure S4), and only the peak of the CH-COOH proton was sensitive to the first deprotonation process at pH < 3, which confirms that pK1 of Pro-SISC-Me applies to COOH. In the presence of 30% (v/v) DMSO only one type of isomer is present (most probably E), or there is a fast exchange process between the isomers (in the case of SISC only one type of isomer was observed in both media).
The pKa of H2L + is only slightly affected by the presence of the DMSO in the case of SISC, while for Pro-SISC a minor decrease is seen. The pKa of HL becomes higher in the case of both ligands, indicating that the HL forms are better solvated in the DMSO/H2O The pK a of H 2 L + is only slightly affected by the presence of the DMSO in the case of SISC, while for Pro-SISC a minor decrease is seen. The pK a of HL becomes higher in the case of both ligands, indicating that the HL forms are better solvated in the DMSO/H 2 O mixture than in pure water. It suggests that the HL form of SISC and Pro-SISC-Me is neutral, as they are shown in Scheme 1. Therefore, the deprotonation of HL is more likely to be assigned to the OH group; however, the proton dissociation processes of H 2 L + and HL are partly overlapped. This is the reason why it is difficult to distinguish between the deprotonation of the phenolic OH and the aminoguanidinium moiety.
Based on the determined pK a values, the proline residue in Pro-SISC-Me is zwitter ionic (COO − , N Pro H + ) in a wide pH range (pH = 4.5-11.5, see the concentration distribution curves in Figure S2c); and its presence in the molecule results in lower pK a values in comparison to those of SISC, especially in the case of pK a of HL (~0.5 log unit), which can be the result of the electron withdrawing effect of the N proline H + moiety and a possible intermolecular hydrogen bond between the deprotonated phenolato oxygen and the N proline H + group.  Based on the determined pKa values, the proline residue in Pro-SISC-Me is zwitter ionic (COO − , NProH + ) in a wide pH range (pH = 4.5-11.5, see the concentration distribution curves in Figure S2c); and its presence in the molecule results in lower pKa values in comparison to those of SISC, especially in the case of pKa of HL (~0.5 log unit), which can be the result of the electron withdrawing effect of the NprolineH + moiety and a possible intermolecular hydrogen bond between the deprotonated phenolato oxygen and the Npro-lineH + group.
Comparing the pKa values of SISC to those of the analogous salicylaldehyde TSC (STSC, pK1 = 8.89; pK2 = 12.59 [22]) and SC (SSC, pK1 = 9.32; pK2 > 12.8 [23]) (Scheme 1) in 30% (v/v) DMSO/H2O, it can be concluded that the exchange of the sulfur in the thioamide moiety of the TSC or the carbonyl oxygen in the SC to the NH group in the aminoguanidine significantly affects the pKa values, thus the actual chemical forms at a given pH. As a consequence, 100% and 97% of SSC and STSC, respectively, are present in their neutral form at pH 7.4; while at this pH, for SISC 64% H2L + and 36% HL and for Pro-SISC-Me 33% H2L + and 67% HL are found. It is noteworthy that HL is neutral but zwitter ionic in the case of Pro-SISC-Me. The different distribution of the species and charges are expected to result in different lipophilicity of the compounds. Distribution coefficients (logD7.4) were determined for the aminoguanidine ligands by n-octanol/water partitioning at pH 7.4 (Table 1, Figure S5). The obtained data for SISC (+0.83) and Pro-SISC-Me (−0.88) are lower than those of STSC (+1.59 [24]) and SSC (+0.94 [23]) due to the presence of the partly positively charged aminoguanidinium moiety and the zwitter ionic amino acid residue in Pro-SISC-Me. Based on these data, the incorporation of the proline residue resulted in a much stronger hydrophilic character as it was also observed e.g., for the proline hybrids of STSC [27], 2-formylpyridine TSC [30] or 8-hydroxyquinolines [33].
It was also found that both compounds possess intrinsic fluorescence at pH 7.4 (see λEX and λEM values in Table 1, and Figure S6) similar to the analogous TSC or SC compounds [22][23][24], and the Pro derivatization resulted in stronger emission intensity. Interestingly, the fluorescence excitation spectrum of Pro-SISC-Me resembles the absorp-  Comparing the pK a values of SISC to those of the analogous salicylaldehyde TSC (STSC, pK 1 = 8.89; pK 2 = 12.59 [22]) and SC (SSC, pK 1 = 9.32; pK 2 > 12.8 [23]) (Scheme 1) in 30% (v/v) DMSO/H 2 O, it can be concluded that the exchange of the sulfur in the thioamide moiety of the TSC or the carbonyl oxygen in the SC to the NH group in the aminoguanidine significantly affects the pK a values, thus the actual chemical forms at a given pH. As a consequence, 100% and 97% of SSC and STSC, respectively, are present in their neutral form at pH 7.4; while at this pH, for SISC 64% H 2 L + and 36% HL and for Pro-SISC-Me 33% H 2 L + and 67% HL are found. It is noteworthy that HL is neutral but zwitter ionic in the case of Pro-SISC-Me. The different distribution of the species and charges are expected to result in different lipophilicity of the compounds. Distribution coefficients (logD 7.4 ) were determined for the aminoguanidine ligands by n-octanol/water partitioning at pH 7.4 (Table 1, Figure S5). The obtained data for SISC (+0.83) and Pro-SISC-Me (−0.88) are lower than those of STSC (+1.59 [24]) and SSC (+0.94 [23]) due to the presence of the partly positively charged aminoguanidinium moiety and the zwitter ionic amino acid residue in Pro-SISC-Me. Based on these data, the incorporation of the proline residue resulted in a much stronger hydrophilic character as it was also observed e.g., for the proline hybrids of STSC [27], 2-formylpyridine TSC [30] or 8-hydroxyquinolines [33].
It was also found that both compounds possess intrinsic fluorescence at pH 7.4 (see λ EX and λ EM values in Table 1, and Figure S6) similar to the analogous TSC or SC compounds [22][23][24], and the Pro derivatization resulted in stronger emission intensity. Interestingly, the fluorescence excitation spectrum of Pro-SISC-Me resembles the absorption spectrum of the L − form and also SISC can be excited at wavelengths (350-400 nm) which are typical for the phenolate form.

Complex Formation of SISC and Pro-SISC-Me with Cu(II) Ions
Interaction between Pro-SISC-Me and Cu(II) ions could be studied by pH-potentiometry in water due to the adequate solubility of the species (Figure S7b), and the calculated overall stability constants are collected in Table 2. Based on the titration data, formation of only mono-ligand complexes ([CuL] + , [CuLH −1 ], [CuLH −2 ] − ) was observed even at higher metal-to-ligand ratios. Stability constants for the complexes formed with the more lipophilic SISC were determined in the presence of 30% (v/v) DMSO/H 2 O ( Figure S7a), and for the Cu(II)-Pro-SISC-Me system was studied as well in the same solvent system for comparison (Table 2). Notably, [CuL] + is formed by the displacement of two protons from the ligand, therefore, coordination of both the phenolato and the aminoguanidine moieties is probable in this species. In the case of SISC, the stability constant could not be obtained for the [CuLH −2 ] − species as in its formation pH range (>8) precipitation appeared in the solution. The thiosemicarbazone analogues (STSC and Pro-STSC-Me) also form exclusively monoligand complexes ( [22,27], however, protonated complexes could not be found for SISC and Pro-SISC-Me.  Complex formation was also monitored by UV-vis spectrophotometric and EPR titrations to confirm the speciation model determined by pH-potentiometry and to reveal the coordination modes. The recorded UV-vis spectra (Figures 4a, S8a and S9a) show characteristic changes upon increasing the pH. Namely, the development of a d-d band with λ max 616 nm was observed in both systems parallel to the formation of the [CuL] + complex, indicating similar coordination modes (Figures 4b and S8b), and the spectra remained intact in a broad pH range (4.5-7.5). Further changes were seen for the Cu(II)-Pro-SISC-Me system in the basic pH range in the wavelength range of both the ligand and d-d bands (Figures 4a and S9a), while in the case of SISC the precipitate formed even at the applied lower concentrations (ca. 0.5 mM, Figure S8a). This finding suggests further changes in the coordination modes, which resemble to the spectral changes reported for the formation of the mixed hydroxido [CuL(OH)] − complexes of Pro-STSC-Me [27]. Thus, complexes formed in the basic pH range are most likely mixed hydroxido species

SISC Pro-SISC-Me
EPR spectra were recorded for the Cu(II)-Pro-SISC-Me species at room temperature in aqueous solution ( Figure 5a) and two-dimensional simulation of the solution EPR spectra resulted in the individual isotropic EPR spectra ( Figure 5c) and parameters (Table S1). Deconvolution of the EPR spectra clearly shows the formation of three different types of complexes: [CuL] + , [CuLH −1 ] and [CuLH −2 ] − , characterized by decreasing g 0 and increasing A 0 parameters. The nitrogen hyperfine splitting, caused by the equatorial coordination of two nitrogen donor atoms, is well-resolved in all component spectra. The two nitrogens were not equivalent, most probably the middle nitrogen (N 1 ) has a higher coupling. No dinuclear or bis-ligand complexes were found under these conditions. The EPR parameters also show the regular elongated octahedral geometry around the Cu(II) ion. Based on these data, we concluded that in [CuL] + , coordination of the ligand via phenolato oxygen, azomethine N and one of the guanidine nitrogens is the most likely (Figure 5b), and the other two species are mixed hydroxido species. It should be noted that hydrogen bonding is possible between the proline nitrogen and the phenolato oxygen as it was found by X-ray crystallography for the Cu(II)-Pro-STSC-Me complex [27].  EPR spectra were recorded for the Cu(II)-Pro-SISC-Me species at room temperature in aqueous solution ( Figure 5a) and two-dimensional simulation of the solution EPR spectra resulted in the individual isotropic EPR spectra ( Figure 5c) and parameters (Table  S1). Deconvolution of the EPR spectra clearly shows the formation of three different types of complexes: [CuL] + , [CuLH−1] and [CuLH−2] − , characterized by decreasing g0 and increasing A0 parameters. The nitrogen hyperfine splitting, caused by the equatorial coordination of two nitrogen donor atoms, is well-resolved in all component spectra. The two nitrogens were not equivalent, most probably the middle nitrogen (N 1 ) has a higher coupling. No dinuclear or bis-ligand complexes were found under these conditions. The EPR parameters also show the regular elongated octahedral geometry around the Cu(II) ion. Based on these data, we concluded that in [CuL] + , coordination of the ligand via phenolato oxygen, azomethine N and one of the guanidine nitrogens is the most likely (Figure 5b), and the other two species are mixed hydroxido species. It should be noted that hydrogen bonding is possible between the proline nitrogen and the phenolato oxygen as it was found by X-ray crystallography for the Cu(II)-Pro-STSC-Me complex [27].
EPR spectra for the Cu(II)-SISC species were recorded at 77 K ( Figure S10a) since the solubility did not allow the measurements at room temperature which requires higher concentrations. All component EPR spectra could be fitted by taking into account rhombic g-and A-tensors (Figure S10b, Table S2) and the superhyperfine splitting of two nitrogens coordinating in the equatorial plane. The [CuL] + complex shows slight rhombic distortion, probably due to the attached chelate rings. The deprotonation of this complex does not affect the coordination sphere and it causes only slight changes in the EPR spectra, similarly as it was seen for the Pro-SISC-Me species. Precipitation occurred at pH > 8 and the spectral intensity decreased, although at pH ca. 12 the solution became clear again owing to the formation of a mixed hydroxido complex ([CuL(OH)2] − ). The superhyperfine lines of this spectra were very well resolved, which made it possible to determine the nitrogen couplings with high accuracy (Table S2). The average of the g parameters of the SISC complexes (g0,calc. in Table S2) were close to those of the directly determined values of the Pro-SISC-Me species, suggesting similar coordination modes in the corresponding complexes. Notably, a dimeric complex of SISC with perchlorate counter ions [(CuLH)2](ClO4)2 was prepared and crystallized out from a methanolic solution by Mondal et al. [3]. The X-ray crystallographic analysis of this complex revealed that the ligands coordinated tridentately via the phenolato O, azomethine N and the amidine N, and the fourth coordination position was occupied by a bridging phenoxide O from the ligand bound to the second Cu(II) of the binuclear unit [3]. However, there was no indication for the presence of such dimeric species in the 30% (v/v) DMSO/H2O solvent mixture based on the EPR spectroscopic measurements. Based on the stability constants, it can be concluded that [CuL] + complexes predominate at pH 7.4 (SISC: 91%, Pro-SISC: 100%). The logD7.4 values were determined for the Cu(II) complexes, and −0.49 ± 0.03 and −0.87 ± 0.03 were obtained for the complex of SISC and Pro-SISC-Me, respectively, revealing their fairly hydrophilic character due to their positive charge. The formation of precipitate in the Cu(II)-SISC system was due to the neutral [CuL(OH)]. For the comparison of the complex stability at pH 7.4, pCu values (the negative decadic logarithm of the equilibrium concentration of the free metal ion, Table 2) were calculated. A higher pCu indicated stronger Cu(II) affinity of the ligand. These values show the negligible dissociation of the complexes under this condition; therefore, they are considered to be a highly stable species and their stability is comparable to that of the tridentate thiosemicarbazone complexes. The Cu(II) binding ability of the proline derivative was somewhat higher in comparison to the reference compound SISC. A similar trend was reported for STSC and Pro-STSC-Me [22,27]. The redox properties of the Cu(II) complexes were investigated as a first step by cyclic voltammetry, and representative voltammograms are shown in Figure S11. Unfortunately, irreversible redox reactions were observed in the pH range of the complex formation, thus, the comparison of these data is not adequate, and prediction of their ability to be reduced by physiological reductants is not possible. Therefore, the direct redox reaction of these Cu(II) complexes with glutathione and ascorbic acid was studied 3000 3200 3400 3600  EPR spectra for the Cu(II)-SISC species were recorded at 77 K ( Figure S10a) since the solubility did not allow the measurements at room temperature which requires higher concentrations. All component EPR spectra could be fitted by taking into account rhombic g-and A-tensors ( Figure S10b, Table S2) and the superhyperfine splitting of two nitrogens coordinating in the equatorial plane. The [CuL] + complex shows slight rhombic distortion, probably due to the attached chelate rings. The deprotonation of this complex does not affect the coordination sphere and it causes only slight changes in the EPR spectra, similarly as it was seen for the Pro-SISC-Me species. Precipitation occurred at pH > 8 and the spectral intensity decreased, although at pH ca. 12 the solution became clear again owing to the formation of a mixed hydroxido complex ([CuL(OH) 2 ] − ). The superhyperfine lines of this spectra were very well resolved, which made it possible to determine the nitrogen couplings with high accuracy (Table S2). The average of the g parameters of the SISC complexes (g 0,calc . in Table S2) were close to those of the directly determined values of the Pro-SISC-Me species, suggesting similar coordination modes in the corresponding complexes. Notably, a dimeric complex of SISC with perchlorate counter ions [(CuLH) 2 ](ClO 4 ) 2 was prepared and crystallized out from a methanolic solution by Mondal et al. [3]. The X-ray crystallographic analysis of this complex revealed that the ligands coordinated tridentately via the phenolato O, azomethine N and the amidine N, and the fourth coordination position was occupied by a bridging phenoxide O from the ligand bound to the second Cu(II) of the binuclear unit [3]. However, there was no indication for the presence of such dimeric species in the 30% (v/v) DMSO/H 2 O solvent mixture based on the EPR spectroscopic measurements.
Based on the stability constants, it can be concluded that [CuL] + complexes predominate at pH 7.4 (SISC: 91%, Pro-SISC: 100%). The logD 7.4 values were determined for the Cu(II) complexes, and −0.49 ± 0.03 and −0.87 ± 0.03 were obtained for the complex of SISC and Pro-SISC-Me, respectively, revealing their fairly hydrophilic character due to their positive charge. The formation of precipitate in the Cu(II)-SISC system was due to the neutral [CuL(OH)]. For the comparison of the complex stability at pH 7.4, pCu values (the negative decadic logarithm of the equilibrium concentration of the free metal ion, Table 2) were calculated. A higher pCu indicated stronger Cu(II) affinity of the ligand. These values show the negligible dissociation of the complexes under this condition; therefore, they are considered to be a highly stable species and their stability is comparable to that of the tridentate thiosemicarbazone complexes. The Cu(II) binding ability of the proline derivative was somewhat higher in comparison to the reference compound SISC. A similar trend was reported for STSC and Pro-STSC-Me [22,27].
The redox properties of the Cu(II) complexes were investigated as a first step by cyclic voltammetry, and representative voltammograms are shown in Figure S11. Unfortunately, irreversible redox reactions were observed in the pH range of the complex formation, thus, the comparison of these data is not adequate, and prediction of their ability to be reduced by physiological reductants is not possible. Therefore, the direct redox reaction of these Cu(II) complexes with glutathione and ascorbic acid was studied at pH 7.4 in 30% (v/v) DMSO/H 2 O solvent mixture by UV-vis spectrophotometry using a tandem cuvette under anaerobic conditions. The mild reducing agent, ascorbate was not able to reduce the complexes. On the other hand, GSH resulted in significant spectral changes in the wavelength range 310-430 nm ( Figure 6). Namely, the decrease of the absorbance at ca. 360 nm was observed, and absorbance was increased at the λ max of the free ligand (316 nm (SISC), 330 nm (Pro-SISC-Me)). It indicates the dissociation of the ligand from the complex upon the reduction, while the forming Cu(I) most probably forms a complex with the GSH that is present in a high excess in the solution. Similar behavior was also reported for the Cu(II) complexes of STSC [24], however, the reaction rate differs. The redox equilibrium was reached within 1 min (SISC), 5 min (Pro-SISC-Me) and 13 min (STSC) under the applied condition (25 µM complex, 250 µM GSH). Therefore, similar to the Cu(II)-STSC complex [24], the complexes of SISC and Pro-SISC-Me are considered as redox active species under physiological relevant conditions, which may contribute to their cytotoxic activity. forms a complex with the GSH that is present in a high excess in the solution. Similar behavior was also reported for the Cu(II) complexes of STSC [24], however, the reaction rate differs. The redox equilibrium was reached within 1 min (SISC), 5 min (Pro-SISC-Me) and 13 min (STSC) under the applied condition (25 µM complex, 250 µM GSH). Therefore, similar to the Cu(II)-STSC complex [24], the complexes of SISC and Pro-SISC-Me are considered as redox active species under physiological relevant conditions, which may contribute to their cytotoxic activity.

Complex Formation of SISC and Pro-SISC-Me with Fe(III) and Fe(II) Ions
As the iron binding ability of the (thio)semicarbazone-related compounds and the difference between the stability of the complexes formed Fe(II) and Fe(III) ions are assumed to have an impact on the biological activity, the stability constants for the complexes formed with SISC and Pro-SISC-Me were determined by the pH-potentiometric titrations in 30% (v/v) DMSO/H2O (Table 3).

Complex Formation of SISC and Pro-SISC-Me with Fe(III) and Fe(II) Ions
As the iron binding ability of the (thio)semicarbazone-related compounds and the difference between the stability of the complexes formed Fe(II) and Fe(III) ions are assumed to have an impact on the biological activity, the stability constants for the complexes formed with SISC and Pro-SISC-Me were determined by the pH-potentiometric titrations in 30% (v/v) DMSO/H 2 O (Table 3).  Complexation with Fe(II) was studied under strictly anaerobic conditions. The obtained titrations' curves (see Figure S12a for the Fe(II)-SISC system) show that the complex formation starts at relatively high pH values (>5.5) with both ligands, and, in the case of SISC precipitate, occurred at pH > 9 at 1:1 and at pH > 10 at 1:2 metal-to-ligand ratios. Evaluation of the titration data revealed that only mono complexes were formed in different protonation states ( Figure S12b) as was also found with the Cu(II) ions (vide supra), although the Fe(II) complexes have a much lower stability. Aside from the better solubility of the Pro-SISC-Me complexes, the two ligands behaved similarly and the difference between their Fe(II)-binding efficacy was minor.

SISC Pro-SISC-Me
Unlike Fe(II), the Fe(III) ions formed bis-ligand complexes as well under the conditions studied. The overall stability constants determined for the [Fe(III)L] 2+ and [Fe(III)L 2 ] + complexes (Table 3) are similar for the two ligands; however, formation of [Fe(III)L 2 H −1 ] and [Fe(III)L 2 H −2 ] − was observed with Pro-SISC-Me at pH > 7 (Figure 7a), while precipitate appeared in this pH range with SISC. In order to clarify this difference, UV-vis spectra were recorded for the Fe(III)-SISC ( Figure S13a) and Fe(III)-Pro-SISC-Me (Figure 8a) systems at various pH values. In the first case, parallel to the formation of the species [Fe(III)L] 2+ a band with λ max = 610 nm appeared and shifted to the lower wavelengths (λ max = 525 nm) as the bis complex was formed, while a strong absorbance decrease was seen at pH > 9 due to precipitation. In the Fe(III)-Pro-SISC-Me system, the formation of the mono complex was accompanied by the development of a band with λ max = 600 nm, while λ max was located at 490 nm as [Fe(III)L 2 ] + was formed, and this band increased up to pH 10.9, but λ max practically remained constant, suggesting that the formation of [Fe(III)L 2 H −1 ] and [Fe(III)L 2 H −2 ] − does not change the type of the coordinated donors. The absorbance was significantly decreased in the strongly basic pH range along with a blue shift, and most probably additional hydrolytic processes took place, e.g., formation of mixed hydroxido species, however for them we could not determine stability constants. The absorbance measured at 490 nm was constant between pH 4.8 and 8.0 (Figure 8a) where [Fe(III)L 2 ] + was formed, and it indicated a different coordination mode compared to the analogous SISC complex (525 nm). In addition, predominance curves calculated for the hypothetic Fe(III)-SISC-Pro-SISC-Me (1:2:2) system (Figure 7b) also exhibited clearly the stronger Fe(III) binding ability of Pro-SISC-Me over SISC, which might be also explained by an altered binding mode.
[Fe(III)L2] + complexes (Table 3) are similar for the two ligands; however, formation of [Fe(III)L2H−1] and [Fe(III)L2H−2] − was observed with Pro-SISC-Me at pH > 7 (Figure 7a), while precipitate appeared in this pH range with SISC. In order to clarify this difference, UV-vis spectra were recorded for the Fe(III)-SISC ( Figure S13a) and Fe(III)-Pro-SISC-Me (Figure 8a) systems at various pH values. In the first case, parallel to the formation of the species [Fe(III)L] 2+ a band with λmax = 610 nm appeared and shifted to the lower wavelengths(λmax = 525 nm) as the bis complex was formed, while a strong absorbance decrease was seen at pH > 9 due to precipitation. In the Fe(III)-Pro-SISC-Me system, the formation of the mono complex was accompanied by the development of a band with λmax = 600 nm, while λmax was located at 490 nm as [Fe(III)L2] + was formed, and this band increased up to pH 10.9, but λmax practically remained constant, suggesting that the formation of [Fe(III)L2H−1] and [Fe(III)L2H−2] − does not change the type of the coordinated donors. The absorbance was significantly decreased in the strongly basic pH range along with a blue shift, and most probably additional hydrolytic processes took place, e.g., formation of mixed hydroxido species, however for them we could not determine stability constants. The absorbance measured at 490 nm was constant between pH 4.8 and 8.0 (Figure 8a) where [Fe(III)L2] + was formed, and it indicated a different coordination mode compared to the analogous SISC complex (525 nm). In addition, predominance curves calculated for the hypothetic Fe(III)-SISC-Pro-SISC-Me (1:2:2) system ( Figure  7b) also exhibited clearly the stronger Fe(III) binding ability of Pro-SISC-Me over SISC, which might be also explained by an altered binding mode.  Further differences were observed when the redox behavior of the iron complexes was monitored by cyclic voltammetry. In the case of SISC, only a cathodic peak could be observed in the pH range of the formation of the Fe(III) complexes (not shown), unlike for Pro-SISC-Me (Figure 8b), where quasi-reversible processes could be seen in a wide Further differences were observed when the redox behavior of the iron complexes was monitored by cyclic voltammetry. In the case of SISC, only a cathodic peak could be observed in the pH range of the formation of the Fe(III) complexes (not shown), unlike for Pro-SISC-Me (Figure 8b), where quasi-reversible processes could be seen in a wide pH range (3.9-10.8). The cathodic peak measured at pH 7.6 was located at much lower potential than in the case of the SISC complex (−0.59 V vs. −0.38 V, Figure S13b). Interestingly, the E 1/2 value of the Fe(III)/Fe(II) redox couple for the Pro-SISC-Me complexes was −0.32 V vs. NHE, which was significantly lower as it was found for the STSC (E 1/2 = −0.14 V vs. NHE) or Triapine (E 1/2 = +0.08 V vs. NHE [34]) complexes. This low redox potential also indicated a strong preference of Pro-SISC-Me for Fe(III) over Fe(II), and it suggested the binding of harder Lewis-base donor atoms than would be expected from the (O,N,N Figure 9 together with some selected bond lengths and angles. The Fe(III) has an elongated octahedral geometry with the tridentate coordination of two meridional ligands. The ligand has two possible coordination sites: the aminoguanidine and the proline part. In literature, there are two crystal structures of Fe(III) with pyridoxal-aminoguanidine (Reference code CELHIM and CELHOS) [35] where the guanidine coordination is realized. Surprisingly, in our case the proline part of the ligands coordinated with the metal ion by the phenolic oxygen, pyrrolidine nitrogen and one carboxylate oxygen. The two tridentate ligands coordinated in a way that the middle (nitrogen) donor atoms occupied the axial positions (Fe1-N5 and Fe1-N10 has somewhat longer bond lengths in comparison with the Fe-O bonds) so that the four oxygens defined the equatorial plane. Owing to the coupled chelate coordination, the octahedron was distorted and the O1-Fe1-O2 and O4-Fe1-O5 angles, similar to the N5-Fe1-N10 deviate from the theoretical 180 • , as they were 167.6(4) • , 164.4(4) • and 164.1(5), respectively. The two phenyl rings turned to the same side of the complex and they were oriented almost parallel to each other (the angle between the ring planes is 8.0(7) • and the distance between the ring centroids was 4.973(9) Å. Due to the relatively large error in the collected data set, the guanidine =NH and -NH 2 groups of the ligands were hardly distinguished, though shorter C-N distances (C8-N3 1.29(2) Å and C23-N9 1.30(2) Å were set to =NH and the longer ones (C8-N4 1.32(2) and C23-N8, 1.31(2) Å) were set to -NH 2 groups. Even the exchange of the two groups in the crystal structure was possible. The crystal lattice was stabilized by C-H . . . O and N-H . . . O bonds between the neighboring ligands, and also through water molecules. The packing arrangement of crystal (2) from a, b and c crystallographic directions are shown in Figure S14 The iron binding properties of SISC and Pro-SISC-Me were compared to structurally related salicylaldehyde TSCs (STSC, Pro-STSC-Me) and Triapine via the calculation of pFe values at pH 7.4 ( Figure 10). Based on these values, the Fe(II)-binding ability of the ligands followed the order: SISC, Pro-SISC-Me < Pro-SSC-Me, STSC << Triapine, while the Fe(III)binding of Triapine was the weakest. The proline hybrids had stronger Fe(III) binding than the reference compounds (SISC, STSC), however, the difference between the pFe(III) and pFe(II) values was the highest for Pro-SISC-Me, most likely due to its (O carboxylate − , N proline , O phenolate − ) binding mode. Since it was reported that the iron preference of the TSCs can influence the cytotoxicity [15,20], the observed differences in the pFe values was expected to affect the bioactivity of these compounds as well.
was stabilized by C-H…O and N-H…O bonds between the neighboring ligands, and also through water molecules. The packing arrangement of crystal (2) from a, b and c crystallographic directions are shown in Figure S14. In all, the coordination of Pro-SISC-Me via the (Ocarboxylate − , Nproline, Ophenolate − ) donor set can explain its different behavior in solution from the reference compound SISC. Most probably this is the binding mode in the [Fe(III)L2] + complex, and [Fe(III)L2H−1] and [Fe(III)L2H−2] − are formed by the deprotonation of the non-coordinating aminoguanidinium moieties. The iron binding properties of SISC and Pro-SISC-Me were compared to structurally related salicylaldehyde TSCs (STSC, Pro-STSC-Me) and Triapine via the calculation of pFe values at pH 7.4 ( Figure 10). Based on these values, the Fe(II)-binding ability of the ligands followed the order: SISC, Pro-SISC-Me < Pro-SSC-Me, STSC << Triapine, while the Fe(III)-binding of Triapine was the weakest. The proline hybrids had stronger Fe(III) binding than the reference compounds (SISC, STSC), however, the difference between the pFe(III) and pFe(II) values was the highest for Pro-SISC-Me, most likely due to its (Ocarboxylate − , Nproline, Ophenolate − ) binding mode. Since it was reported that the iron preference of the TSCs can influence the cytotoxicity [15,20], the observed differences in the pFe values was expected to affect the bioactivity of these compounds as well.

In Vitro Cytotoxicity of the Compounds
The cytotoxic activity of SISC and Pro-SISC-Me was assayed in the chemo-sensitive Colo-205, and the doxorubicin-resistant Colo-320 human colon adenocarcinoma cell lines, using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Additionally, the cytotoxicity was measured in normal human embryonal lung fibroblast cells (MRC-5). Determined IC50 values using 72 h incubation time are collected in Table 4. Measurements were also performed in the presence of one equivalent Cu(II) ions and comparative data for STSC and Triapine are also shown (Table 4).

In Vitro Cytotoxicity of the Compounds
The cytotoxic activity of SISC and Pro-SISC-Me was assayed in the chemo-sensitive Colo-205, and the doxorubicin-resistant Colo-320 human colon adenocarcinoma cell lines, using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Additionally, the cytotoxicity was measured in normal human embryonal lung fibroblast cells (MRC-5). Determined IC 50 values using 72 h incubation time are collected in Table 4. Measurements were also performed in the presence of one equivalent Cu(II) ions and comparative data for STSC and Triapine are also shown (Table 4). Based on the IC 50 values, ligands SISC and Pro-SISC-Me are not cytotoxic, STSC has fairly weak cytotoxicity, and only Triapine is characterized by IC 50 values in the low µM concentrations. The lower activity of SISC, Pro-SISC-Me and STSC can be explained by their inability to bind iron efficiently in both +2 and +3 oxidation states (see the pFe values in Figure 10), which seems to be necessary for biological activity and the iron complexes of these ligands cannot go through redox cycling between the two oxidation states. However, this is unlike Triapine, which acts as an efficient Fe(II) binder as well and is characterized by a moderate (and not too negative) redox potential (as is explained in the previous section).
In the presence of Cu(II) ions, SISC and STSC displayed more significant anticancer activity than the ligands alone, while the Triapine became less active. Pro-SISC-Me remained non-toxic upon complexation with Cu(II). The complex of STSC is neutral and lipophilic at pH 7.4 (logD 7.4 = +1.14 [24]) which most probably accounts for the enhanced cytotoxic activity. The Cu(II) complex of SISC is more hydrophilic (logD 7.4 = −0.49 ± 0.03) as it is present mostly in its positively charged form at pH 7.4 (90% [CuL] + , 10% [CuL(OH)]), and this feature can result in a smaller increase in the cytotoxicity, however, the complex is redox-active, based on our measurement. The Cu(II) complex of SISC was found to be cytotoxic against the human colon cancer cell line HCT16 as well as showing apoptosis induction [3].
In the case of Pro-SISC-Me, the [CuL] + form also predominates, which contains the zwitter ionic (COO − , N Pro H + ) moiety and as a result it is even more hydrophilic (logD 7.4 = −0.87 ± 0.03), which is not advantageous for an efficient cellular uptake. The Triapine Cu(II) complex is also positively charged at pH 7.4 [21].

Chemicals
STSC, KCl, HCl, KOH, KNO 3 , DMSO, DMF, KSCN, tetrabutylammonium nitrate (TBAN), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 4,4-dimethyl-4silapentane-1-sulfonic acid (DSS) were purchased from Sigma-Aldrich (Budapest, Hungary) in puriss quality. The Fe(II) stock solution was obtained from fine Fe powder dissolved in a known amount of HCl solution under a purified, strictly oxygen-free argon atmosphere, then filtered, stored and used under anaerobic conditions. KSCN solution was used to check the absence of Fe(III) traces in the Fe(II) solution. The concentration of the Fe(II) stock solution was determined by permanganometric titrations under acidic conditions. FeCl 3 and anhydrous CuCl 2 were dissolved in known amounts of HCl and in water, respectively, in order to get the Fe(III) and Cu(II) stock solutions. Their concentrations were determined by complexometry, via the EDTA complexes. The strong acid content of the metal stock solutions were determined by pH-potentiometric titrations. All solvents were of analytical grade and used without further purification. Milli-Q water was used for sample preparation.

Synthesis of SISC and Pro-SISC-Me
SISC·HCl·0.5C 2 H 5 OH·H 2 O was prepared as reported in the literature [5]. For the preparation of Pro-SISC-Me·HCl·H 2 O, a solution of aminoguanidine bicarbonate (0.52 g, 3.8 mmol) in hot water (1 mL) treated with 2 equiv. 6 M HCl was added to a warm solution of the (2S)-1-[(3-formyl-2-hydroxy-5-methylphenyl)methyl]-pyrrolidine-2-carboxylic acid (1.00 g, 3.8 mmol) in methanol (5 mL). The reaction mixture was stirred at 75 • C for 2 h. After cooling the solution to room temperature, methanol was removed under reduced pressure and water (2 mL) was added. The white precipitate was filtered off, washed with water and dried in vacuo overnight. Yield: 0.91 g (61%). Calcd. for C 15  Single crystals suitable for X-ray diffraction experiment of 1 were picked up from the synthesized product, while single crystals of 2 were grown from methanol. The solution contained FeCl 3 and Pro-SISC-Me at 1:2 metal-to-ligand ratio and NaOH was added step by step until the green solution turned into red. A red, single crystal was mounted on a loop and transferred to the goniometer.
X-ray diffraction data for 1 and 2 were collected on a Bruker X8 APEXII CCD and Rigaku RAXIS-RAPID II diffractometer, respectively. The data were processed using SAINT software [36] for 1 and CrystalClear software [37] for 2. Crystal data, data collection parameters, and structure refinement details for 1 and 2 are given in Tables S3 and S4, respectively. Both structures were solved by direct methods and refined by full-matrix least-squares techniques. Non-hydrogen atoms were refined with anisotropic displacement parameters. H atoms were inserted into calculated positions and refined with a riding model, even though their positions could be determined in the case of 1 by a difference Fourier map.
The following computer programs and hardware were used for 1: structure solution: SHELXS; refinement: SHELXL [38]; molecular diagrams: ORTEP [39]; computer: Intel CoreDuo. In the case of 2, Sir2014 [40] and SHELXL [38] under WinGX [41] software were used for structure solution and refinement, respectively. Due to the poor crystal quality and small crystal size of 2, high R values were obtained which prevented the determination of hydrogen atomic positions from the difference Fourier map. Therefore, hydrogen atoms were included in the structure factor calculations, but they were not refined. The isotropic displacement parameters of the hydrogen atoms were approximated from the U(eq) value of the atom they were bonded to. Water hydrogens were fixed in geometric positions using DFIX and DANG options to obtain reasonable positions for them. Selected bond lengths and angles of compounds were calculated by PLATON software [42].
The crystallographic data files for the compounds have been deposited with the Cambridge Crystallographic Database as CCDC 2143435 (1) and CCDC 2144192 (2).

pH-Potentiometry
The pH-potentiometric measurements for the determination of the proton dissociation constants of the ligands and the overall stability constants of the metal complexes were carried out at 25.0 ± 0.  [43]. The average water ionization constant pK w was 13.76 ± 0.05 (water) or 14.52 ± 0.05 (30% (v/v) DMSO/H 2 O). The pH-potentiometric titrations were performed in the pH range 2.0-12.5. The initial volume of the samples was 10.0 mL. The ligand concentration was 2 mM and metal ion-to-ligand ratios of 1:1-1:3 were used. The accepted fitting of the titration curves was always less than 0.01 mL. Argon was always passed over the solutions during the titrations. The exact concentration of the ligand stock solutions together with the proton dissociation constants were determined by pHpotentiometric titrations with the use of the computer program HYPERQUAD [44]. It was also utilized to establish the stoichiometry of the complexes and to calculate the stability constants (β(M p L q H r )). β(M p L q H r ) is defined for the general equilibrium pM + qL + rH ionic strength of 0.10 M (KCl). The titrations wer solution of known concentration (0.10 M). The co were determined by pH-potentiometric titrations with a Metrohm combined electrode (type 6.0234. rette were used for the titrations. The electrode −log[H+] scale by means of blank titrations (stron according to the method suggested by Irving et constant pKw was 13.76 ± 0.05 (water) or 14.52 pH-potentiometric titrations were performed in th of the samples was 10.0 mL. The ligand concentrat ratios of 1:1-1:3 were used. The accepted fitting o than 0.01 mL. Argon was always passed over the act concentration of the ligand stock solutions tog stants were determined by pH-potentiometric tit program HYPERQUAD [44]. It was also utilized complexes and to calculate the stability constants the general equilibrium pM + qL + rH ⇋ MpLqHr where M denotes the metal ion and L the complet tions exclusively, titration data were used from ex visible in the reaction mixture.

UV-Vis Spectrophotometry and Fluorometry
An Agilent Cary 8454 diode array spectroph Clara, CA, USA) was used to record the UV-vis sp

UV-Vis Spectrophotometry and Fluorometry
An Agilent Cary 8454 diode array spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) was used to record the UV-vis spectra at an interval of 200-800 nm. The path length was 1 or 2 cm. Spectrophotometric titrations were performed in water or in 30% (v/v) DMSO/H 2 O on samples containing the ligands at 50 µM-1 mM concentration, in the pH range from 1.0 to 12.5 in the absence or in the presence of 1 or 0.5 or 0.33 equiv. metal ions. K a values of the ligands and the UV-vis spectra of the individual species were calculated by the computer program PSEQUAD [45].
The redox reaction of the Cu(II) complexes with GSH and ascorbic acid was studied in 30% (v/v) DMSO/H 2 O at 25.0 ± 0.1 • C using a special, tightly closed tandem cuvette (Hellma Tandem Cell, 238-QS (Hellma Materials GmbH, Jena, Germany)). The reactants were separated until the reaction was triggered. Both isolated pockets of the cuvette were completely deoxygenated by bubbling of a stream of Ar for 10 min before mixing the reactants. Spectra were recorded before and then immediately after the mixing, and changes were followed until no further absorbance change was observed. One of the isolated pockets contained the reducing agent, while the other contained the metal complex, and their final concentrations after mixing were 250 µM and 25 µM, respectively. The pH of all the solutions was adjusted to 7.40 by 50 mM HEPES buffer and an ionic strength of 0.1 M (KCl) was applied. The stock solutions of the reducing agents and the complexes were freshly prepared every day.
The fluorescence spectra of the ligands were recorded on a Hitachi F-4500 spectrofluorometer in water at pH 7.4 (Hitachi High-Technologies Corporation, Tokyo, Japan) using a 1 × 1 cm quartz cell.

1 H NMR Spectroscopic Titrations
The 1 H NMR studies for the ligands were carried out on a Bruker Ultrashield 500 Plus instrument (Billerica, MA, USA). DSS was used as an internal NMR standard and WA-TERGATE water suppression pulse scheme was used. Spectra were recorded in 10% (v/v) D 2 O/H 2 O or in 30% (v/v) DMSO-d 6 /H 2 O solvent mixtures in a concentration of 1 mM, respectively, at ionic strength of 0.10 M (KCl).

EPR Spectroscopy
All EPR spectra were recorded with a BRUKER EleXsys E500 spectrometer (microwave frequency 9.54 GHz, microwave power 13 mW, modulation amplitude 5 G, modulation frequency 100 kHz, Bruker BioSpin Corporation, Billerica, MA, USA) at 77 K for the Cu(II)-SISC complexes and at room temperature for the Cu(II)-Pro-SISC-Me species. EPR spectra were recorded at different pH values at two metal-to-ligand concentration ratios: 0.25 mM Cu(II) and 0.25 mM or 0.50 mM SISC in 30% DMSO/water solution, and 1.0 mM Pro-SISC-Me and 1.0 mM or 0.5 mM Cu(II) in water. The ionic strength was adjusted with KCl (I = 0.1 M). KOH solution was added to the stock solution to change the pH between pH = 2.5-12.5, which was measured with a Radiometer PHM240 pH/ion Meter equipped with a Metrohm 6.0234.100 glass electrode. The isotropic EPR spectra were recorded at room temperature in a circulating system, and a Heidolph Pumpdrive 5101 peristaltic pump was used to circulate the solution from the titration vessel through a capillary tube into the cavity of the instrument. For the anisotropic EPR spectra, a pH titration was performed outside of the cavity and at selected pH values, and 200 µL samples were transferred from the titration vessel into EPR tubes and the spectra were recorded in a Dewar containing liquid nitrogen.
The anisotropic EPR spectra were analyzed with the EPR program [46]. Rhombic g-tensor (g x, g y , g z ) and copper hyperfine tensor (A x , A y , A z ) have been used. The nitrogen superhyperfine structure was taken into account with a rhombic hyperfine tensor (a x N , a y N , a z N ), where the xyz directions referred to the g-tensor orientations. Orientation-dependent linewidth parameters (α, β, and γ) were used to fit the linewidths through the equation σ MI = α + βM I + γM I 2 , where M I denotes the magnetic quantum number of Cu(II) ion. Since natural CuCl 2 was used for the measurements, the spectra were calculated by the summation of spectra 63 Cu and 65 Cu, weighted by their natural abundances. The hyperfine and superhyperfine coupling constants and the relaxation parameters were obtained in field units (Gauss = 10 −4 T). The series of room temperature EPR spectra were simulated simultaneously by the 'two-dimensional' method using the 2D_EPR program [47]. Each component curve was described by the isotropic EPR parameters g 0 , A 0 copper hyperfine and A 0 N nitrogen hyperfine couplings and isotropic linewidth parameters (α, β, and γ). The concentrations of the complexes were varied by fitting their formation constants β(M p L q H r ). The details of the analysis were published previously [20,22,27,30].

Cyclic Voltammetry
Cyclic voltammograms of the Cu(II) and Fe(III) complexes in 30% (v/v) DMSO/H 2 O containing 1 mM Cu(NO 3 ) 2 or 1 mM or 0.5 mM FeCl 3 and 1 mM ligand were recorded at 25.0 ± 0.1 • C. Ionic strength was 0.10 M (TBAN). Measurements were performed on a conventional three-electrode system under nitrogen atmosphere using an Autolab PGSTAT 204 potentiostat/galvanostat monitored by Metrohm's Nova software (Metrohm Autolab B.V., Utrecht, The Netherlands). Samples were purged with argon for 15 min before recording the cyclic voltammograms. Platinum electrodes were used as the working and auxiliary electrode and Ag/AgCl/3 M KCl as a reference electrode. The electrochemical system was calibrated with an aqueous solution of K 3 [Fe(CN) 6 ] (E 1/2 = +0.386 V vs. NHE).

In Vitro Cell Studies: Cell Lines and Culture Conditions and MTT Assay
All cell culture reagents were obtained from Sigma-Aldrich and plastic ware from Sarstedt (Nümbrecht, Germany). Human colon Colo-205 (chemo-sensitive) and Colo-320 The tested compounds were dissolved in 90% (v/v) DMSO/H 2 O using 8 mM concentration, and the and in the final samples the DMSO content was always lower than 1%. Then stock solutions were diluted in complete culture medium, and two-fold serial dilutions of compounds were prepared in 100 µL of the medium, horizontally. The semi-adherent colon adenocarcinoma cells were treated with Trypsin-Versene (EDTA) solution. They were adjusted to a density of 1 × 10 4 cells in 100 µL of RPMI 1640 or EMEM medium and were added to each well, with the exception of the medium control wells. The final volume of the wells containing compounds and cells was 200 µL. The plates containing Colo-205, Colo-320 or MRC-5 cells were incubated at 37 • C for 72 h; at the end of the incubation period, 20 µL of MTT solution (from a stock solution of 5 mg/mL) were added to each well. After incubation at 37 • C for 4 h, 100 µL of SDS solution (10% in 0.01 M HCI) were added to each well, and the plates were further incubated at 37 • C overnight. Cell growth was determined by measuring the optical density (OD) at 540/630 nm with a Multiskan EX plate reader (Thermo Labsystems, Cheshire, WA, USA). Inhibition of the cell growth (expressed as IC 50 : inhibitory concentration that reduces by 50% the growth of the cells exposed to the tested compounds) was determined from the sigmoid curve where 100 − ((OD sample − OD medium control )/(OD cell control − OD medium control )) × 100 values were plotted against the logarithm of compound concentrations. Curves were fitted by GraphPad Prism software (2021, Graphpad Software, San Diego, CA, USA) [48] using the sigmoidal dose-response model (comparing variable and fixed slopes). The IC 50 values were obtained from at least three independent experiments.

Conclusions
Two salicylaldehyde aminoguanidine derivatives (SISC, Pro-SISC-Me) were prepared as salicylaldehyde thiosemicarbazone analogues to investigate the effect of the exchange of the thioamide sulfur (=S) to an iminosemicarbazone (=NH) moiety on the pK a values and lipophilic character, the complex formation ability with endogenous metal ions such as Cu(II), Fe(II) and Fe(III) and their cytotoxic activity. Pro-SISC-Me contains an additional L-proline moiety to provide enhanced water solubility due to its zwitter ionic structure.
The proton dissociation processes were characterized by the combined use of pHpotentiometry, UV-vis and 1 H NMR spectroscopic titrations in 30% (v/v) DMSO/H 2 O and in water. SISC was characterized by two pK a values which belong to the overlapping deprotonation processes of the phenolic-OH and the positively charged aminoguanidinium (C(=NH 2 + )NH 2 ) functional groups. Pro-SISC-Me has the additional pK a of the proline carboxylic acid group, while the proline nitrogen remains protonated in the whole pH range studied. Based on the pK a values, SISC is found at pH 7.4 in its H 2 L + (64%) and HL (36%) forms, while the analogous STSC is neutral (H 2 L), and Pro-SISC-Me is present in its H 2 L + (33%) and HL (67%) forms which contain the zwitter ionic (N Pro H + , COO − ) amino acid residue contributing to its considerably hydrophilic character.
The stability of the Cu(II) complexes was characterized by pH-potentiometry, and the speciation model was confirmed by UV-vis measurements. Mono-ligand complexes ([CuL] + , [CuLH −1 ], [CuLH −2 ] − ) were found exclusively even at ligand excess. The Cu(II) complexes of both SISC and Pro-SISC-Me possess high stability in solution, which is comparable to that of the related thiosemicarbazones: STSC and Pro-STSC-Me. Complex [CuL] + predominates at pH 7.4 in which the ligands coordinate in a tridentate mode via the phenolato oxygen, azomethine-N and one of the guanidine nitrogens based on the EPR spectroscopic parameters. Complexes [CuLH −1 ] and [CuLH −2 ] − are mixed hydroxido species. The EPR spectroscopic data suggest the same coordination modes in the corresponding SISC and Pro-SISC-Me complexes. The [CuL] + complexes could not be reduced by ascorbate at pH 7.4, whereas they could oxidize GSH efficiently.
Unlike TSCs, SISC and Pro-SISC form mono complexes with Fe(II) exclusively under the applied conditions, however their stability, which is otherwise similar to each other, is much lower compared to that of the Cu(II) complexes. On the contrary, bis complexes are also formed with Fe(III) ions and complex [Fe(III)L 2 ] + is found at pH 7.4 in solution.
Interestingly, [Fe(III)L 2 ] + formed with Pro-SISC-Me has significantly higher stability in comparison to the SISC complex. Complexes [Fe(III)L 2 ] + have different structures also suggested by their different λ max (Pro-SISC-Me: 490 nm, SISC: 525 nm) and redox potential (Pro-SISC-Me: E 1/2 = −0.32 V vs. NHE, SISC: only a cathodic peak at −0.17 V vs. NHE). In the case of Pro-SISC-Me, the proline moiety is involved in the coordination and the ligands bind in the bis complex via the (O carboxylate − , N Pro , O phenolate − ) donor set confirmed by X-ray crystallographic analysis. Fe(III) ions form complexes that possess higher stability with both ligands than Fe(II), although, the (O carboxylate − , N Pro , O phenolate − ) binding mode brings the even stronger Fe(III) preference over Fe(II) of Pro-SISC-Me compared to SISC. Most likely, this feature contributes to the lack of cytotoxic activity of SISC and Pro-SISC. It is worth noting that the redox active Cu(II) complex of SISC displayed a higher cytotoxic activity than the ligand alone, whereas the Cu(II) complex of Pro-SISC-Me was inactive, most probably due to its fairly hydrophilic nature.